Search

We previously identified a lipopeptide, EK1C4, by linking cholesterol to EK1, a pan-CoV fusion inhibitory peptide via a polyethylene glycol (PEG) linker, which showed potent pan-CoV fusion inhibitory activity. However, PEG can elicit antibodies to PEG in vivo, which will attenuate its antiviral activity. Therefore, we designed and synthesized a dePEGylated lipopeptide, EKL1C, by replacing the PEG linker in EK1C4 with a short peptide. Similar to EK1C4, EKL1C displayed potent inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses. In this study, we found that EKL1C also exhibited broad-spectrum fusion inhibitory activity against human immunodeficiency virus type 1 (HIV-1) infection by interacting with the N-terminal heptad repeat 1 (HR1) of viral gp41 to block six-helix bundle (6-HB) formation. These results suggest that HR1 is a common target for the development of broad-spectrum viral fusion inhibitors and EKL1C has potential clinical application as a candidate therapeutic or preventive agent against infection by coronavirus, HIV-1, and possibly other class I enveloped viruses.

The emergence of SARS-CoV-2 variants and potentially other highly pathogenic sarbecoviruses in the future highlights the need for pan-sarbecovirus vaccines. Here, we discovered a new STING agonist, CF501, and found that CF501-adjuvanted RBD-Fc vaccine (CF501/RBD-Fc) elicited significantly stronger neutralizing antibody (nAb) and T cell responses than Alum- and cGAMP-adjuvanted RBD-Fc in mice. Vaccination of rabbits and rhesus macaques (nonhuman primates, NHPs) with CF501/RBD-Fc elicited exceptionally potent nAb responses against SARS-CoV-2 and its nine variants and 41 S-mutants, SARS-CoV and bat SARSr-CoVs. CF501/RBD-Fc-immunized hACE2-transgenic mice were almost completely protected against SARS-CoV-2 challenge, even 6 months after the initial immunization. NHPs immunized with a single dose of CF501/RBD-Fc produced high titers of nAbs. The immunized macaques also exhibited durable humoral and cellular immune responses and showed remarkably reduced viral load in the upper and lower airways upon SARS-CoV-2 challenge even at 108 days post the final immunization. Thus, CF501/RBD-Fc can be further developed as a novel pan-sarbecovirus vaccine to combat current and future outbreaks of sarbecovirus diseases.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmission rates and striking immune evasion have posed a serious challenge to the application of current first-generation SARS-CoV-2 vaccines. Other sarbecoviruses, such as SARS-CoV and SARS-related coronaviruses (SARSr-CoVs), have the potential to cause outbreaks in the future. These facts call for the development of variant-proof SARS-CoV-2, pan-sarbecovirus or pan-β-CoV vaccines. Several novel vaccine platforms have been used to develop vaccines with broad-spectrum neutralizing antibody responses and protective immunity to combat the current SARS-CoV-2 and its variants, other sarbecoviruses, as well as other β-CoVs, in the future. In this review, we discussed the major target antigens and protective efficacy of current SARS-CoV-2 vaccines and summarized recent advances in broad-spectrum vaccines against sarbecoviruses and β-CoVs.

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.

The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases., Graphical abstract The dePEGylated lipopeptides were designed, synthesized, and screened for the most effective one, EKL1C, which was shown to have broad-spectrum anti-coronavirus activity, potent protection of human angiotensin-converting enzyme 2 (hACE2) transgenic mice against authentic coronavirus infection, and high stability.Image 1

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, causes immunosuppression and high mortality in 3–6-week-old chickens. Innate immune defense is a physical barrier to restrict viral replication. After viral infection, the host shows crucial defense responses, such as stimulation of antiviral effectors to restrict viral replication. Here, we conducted RNA-seq in avian cells infected by IBDV and identified TRIM25 as a host restriction factor. Specifically, TRIM25 deficiency dramatically increased viral yields, whereas overexpression of TRIM25 significantly inhibited IBDV replication. Immunoprecipitation assays indicated that TRIM25 only interacted with VP3 among all viral proteins, mediating its K27-linked polyubiquitination and subsequent proteasomal degradation. Moreover, the Lys854 residue of VP3 was identified as the key target site for the ubiquitination catalyzed by TRIM25. The ubiquitination site destroyed enhanced the replication ability of IBDV in vitro and in vivo. These findings demonstrated that TRIM25 inhibited IBDV replication by specifically ubiquitinating and degrading the structural protein VP3., Author summary Owing to the immunosuppression and high mortality caused by infectious bursal disease virus (IBDV) in chickens, hosts urgently boost their antiviral immune responses to resist IBDV infection. Here, we identified the host antiviral factor TRIM25, which defended against IBDV infection by direct targeting the viral protein VP3 for subsequent ubiquitination and degradation. To our knowledge, this is the first report of host TRIM25 restricting IBDV replication. Additionally, we found that Lys854 of VP3 was the target site ubiquitinated by TRIM25. Mutation in the ubiquitination site of VP3 abrogated the degradation of VP3 and further enhanced IBDV replication, but did not influence its virulence. Our work not only uncovers an all-important molecular mechanism of chicken TRIM25 against IBDV but also provides new insights for developing high-titer live IBDV vaccines.

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na(+)/H(+) exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps. IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Since subgroup J avian leukosis virus (ALV-J) was first isolated in the United Kingdom in 1988, it has seriously hindered the development of the poultry industry worldwide. Although cases of ALV-J infection have been reported as early as 2001 in Pakistan, there was no further research on the isolation and molecular characteristics of ALVs. In the present study, we first isolated two ALVs from suspicious clinical samples that were collected from a desi chicken farm in Pakistan. The results of multiplex PCR and indirect immunofluorescent antibody assays confirmed that the two isolates (PK19FA01 and PK19SA01) belonged to ALV-J. The complete genomes of the two isolates were amplified, sequenced, and systematically analyzed. We found that gp85 of PK19FA01 was more similar to that of the prototype strain HPRS103, whereas gp85 of PK19SA01 was more similar to that of American strains. The two isolates contained an intact E element of 147 residues and had a unique 135 bp deletion in the redundant transmembrane of the 3′ untranslated region. The U3 region of the two isolates was highly homologous to that of American ALV-J strains. To our knowledge, this is the first report of the isolation, complete genome sequencing, and systematic molecular epidemiological investigation of ALV-J in Pakistan. Our findings could enrich epidemiological data and might contributed to more effective measures to prevent and control avian leukosis in Pakistan.

Avian leukosis virus subgroup J (ALV-J) was first isolated from broiler chickens in China in 1999; subsequently, it was rapidly introduced into layer chickens and Chinese local chickens. Recently, the incidence of ALV-J in broiler and layer chickens has significantly decreased. However, it has caused substantial damage to Chinese local chickens, resulting in immense challenges to their production performance and breeding safety. To systematically analyze the molecular characteristics and the epidemic trend of ALV-J in Chinese local chickens, 260 clinical samples were collected for the period of 2013–2018; 18 ALV-J local chicken isolates were identified by antigen-capture enzyme-linked immunosorbent assay and subgroup A-, B-, and J-specific multiplex PCR. The whole genomic sequences of 18 isolates were amplified with PCR and submitted to GenBank. Approximately, 55.5% (10/18) of the 18 isolates demonstrated a relatively high homology (92.3–95.4%) with 20 ALV-J early-isolated local strains (genome sequences obtained from GenBank) in gp85 genes clustering in a separated branch. The 3ʹ untranslated region (3ʹ UTR) of the 18 isolates showed a 195–210 and 16–28 base pair deletion in the redundant transmembrane region and in direct repeat 1, respectively; 55.5% (10/18) of the 18 isolates retained the 147 residue E element. The U3 gene of 61.1% (11/18) of the 18 isolates shared high identity (94.6–97.3%) with ALV-J early-isolated local strains. These results implied that the gp85 and U3 of ALV-J local chicken isolates have rapidly evolved and formed a unique local chicken branch. In addition, it was determined that the gene deletion in the 3′UTR region currently serves as a unique molecular characteristic of ALV-J in China. Hence, the obtained results built on the existing ALV-J molecular epidemiological data and further elucidated the genetic evolution trend of ALV-J in Chinese local chickens.

Avian leukosis virus subgroup J (ALV-J), a highly oncogenic retrovirus, causes leukemia-like proliferative diseases in chickens. microRNAs post-transcriptionally suppress targets and are involved in the development of various tumors. We previously showed that miR-221 is upregulated in ALV-J-induced tumors. In this study, we analyzed the possible function of miR-221 in ALV-J tumorigenesis. The target validation system showed that CDKN1B is a target of miR-221 and is downregulated in ALV-J infection. As CDKN1B arrests the cell cycle and regulates its progression, we analyzed the proliferation of ALV-J-infected DF-1 cells. ALV-J-infection-induced DF1 cell derepression of G1/S transition and overproliferation required high miR-221 expression followed by CDKN1B downregulation. Cell cycle pathway analysis showed that ALV-J infection induced DF-1 cell overproliferation via the CDKN1B-CDK2/CDK6 pathway. Thus, miR-221 may play an important role in ALV-J-induced aggressive growth of DF-1 cells; these findings have expanded our insights into the mechanism underlying ALV-J infection and tumorigenesis.

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3&prime, untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25 ng/ml for p27 protein and 80 TCID50/ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.